New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting.,Analytical Chemistry

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New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-04-10 , DOI: 10.1021/acs.analchem.0c00680
Gaoting Wang ₁ , Weimin Tian ₁ , Xiaoling Liu ₁ , Wei Ren ₁ , Chenghui Liu ₁

Affiliation

Current CRISPR-Cas-based nucleic acid sensing methods relying on the preassembled Cas-crRNA complexes are generally limited to the detection of protospacer-adjacent motif (PAM)-containing sequences, and nonspecific backgrounds are inevitable. Herein, we propose a new CRISPR-derived microRNA sensing mechanism based on rolling circle transcription (RCT)-unleashed self-recruiting of crRNA by Cas12a (Cas12a-SCR). In Cas12a-SCR, target microRNA can specifically trigger RCT to produce a long single-strand RNA with numerous pre-crRNA repeats, which can be trimmed and recruited by Cas12a actively. This new target-initiated, real-time producing, trimming, and self-assembling manner of Cas12a-crRNA remarkably suppresses the nonspecific background and relieves the stringent requirement of PAM site in the target sequence. Thus, the universality of the Cas12a-SCR toward different nucleic acid sequences is greatly expanded.

中文翻译：

基于Cas12a自供电和滚动圈转录释放实时crRNA募集的CRISPR衍生的新microRNA传感机制。

当前依赖于预组装的Cas-crRNA复合物的基于CRISPR-Cas的核酸传感方法通常仅限于检测含有原间隔物的相邻基序（PAM）的序列，并且非特异性背景是不可避免的。在本文中，我们提出了一种新的基于CRISPR的microRNA传感机制，该机制基于Cas12a（Cas12a-SCR）对crRNA进行滚动圈转录（RCT）释放的自招募。在Cas12a-SCR中，目标微RNA可以特异性地触发RCT，以产生具有许多pre-crRNA重复序列的长单链RNA，Cas12a可以主动对其进行修剪和募集。Cas12a-crRNA的这种新的靶标起始，实时产生，修饰和自组装方式显着抑制了非特异性背景并减轻了靶标序列中PAM位点的严格要求。从而，

更新日期：2020-04-10

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